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1.  A Noncompetitive Inhibitor for Mycobacterium tuberculosis's Class IIa Fructose 1,6-Bisphosphate Aldolase 
Biochemistry  2013;53(1):202-213.
Class II fructose 1,6-bisphosphate aldolase (FBA) is an enzyme critical for bacterial, fungal, and protozoan glycolysis/gluconeogenesis. Importantly, humans lack this type of aldolase, having instead a class I FBA that is structurally and mechanistically distinct from class II FBAs. As such, class II FBA is considered a putative pharmacological target for the development of novel antibiotics against pathogenic bacteria such as Mycobacterium tuberculosis, the causative agent for tuberculosis (TB). To date, several competitive class II FBA substrate mimic-styled inhibitors have been developed; however, they lack either specificity, potency, or properties that limit their potential as possible therapeutics. Recently, through the use of enzymatic and structure-based assisted screening, we identified 8-hydroxyquinoline carboxylic acid (HCA) that has an IC50 of 10 ± 1 μM for the class II FBA present in M. tuberculosis (MtFBA). As opposed to previous inhibitors, HCA behaves in a noncompetitive manner, shows no inhibitory properties toward human and rabbit class I FBAs, and possesses anti-TB properties. Furthermore, we were able to determine the crystal structure of HCA bound to MtFBA to 2.1 Å. HCA also demonstrates inhibitory effects for other class II FBAs, including pathogenic bacteria such as methicillin-resistant Staphylococcus aureus. With its broad-spectrum potential, unique inhibitory characteristics, and flexibility of functionalization, the HCA scaffold likely represents an important advancement in the development of class II FBA inhibitors that can serve as viable preclinical candidates.
doi:10.1021/bi401022b
PMCID: PMC4167715  PMID: 24325645
2.  Long-term Survival and Virulence of Mycobacterium leprae in Amoebal Cysts 
Leprosy is a curable neglected disease of humans caused by Mycobacterium leprae that affects the skin and peripheral nerves and manifests clinically in various forms ranging from self-resolving, tuberculoid leprosy to lepromatous leprosy having significant pathology with ensuing disfiguration disability and social stigma. Despite the global success of multi-drug therapy (MDT), incidences of clinical leprosy have been observed in individuals with no apparent exposure to other cases, suggestive of possible non-human sources of the bacteria. In this study we show that common free-living amoebae (FLA) can phagocytose M. leprae, and allow the bacillus to remain viable for up to 8 months within amoebic cysts. Viable bacilli were extracted from separate encysted cocultures comprising three common Acanthamoeba spp.: A. lenticulata, A. castellanii, and A. polyphaga and two strains of Hartmannella vermiformis. Trophozoites of these common FLA take up M. leprae by phagocytosis. M. leprae from infected trophozoites induced to encyst for long-term storage of the bacilli emerged viable by assessment of membrane integrity. The majority (80%) of mice that were injected with bacilli extracted from 35 day cocultures of encysted/excysted A. castellanii and A. polyphaga showed lesion development that was similar to mice challenged with fresh M. leprae from passage mice albeit at a slower initial rate. Mice challenged with coculture-extracted bacilli showed evidence of acid-fast bacteria and positive PCR signal for M. leprae. These data support the conclusion that M. leprae can remain viable long-term in environmentally ubiquitous FLA and retain virulence as assessed in the nu/nu mouse model. Additionally, this work supports the idea that M. leprae might be sustained in the environment between hosts in FLA and such residence in FLA may provide a macrophage-like niche contributing to the higher-than-expected rate of leprosy transmission despite a significant decrease in human reservoirs due to MDT.
Author Summary
Leprosy is a progressive disease of the skin and nervous system caused by the bacillus, Mycobacterium leprae. Implementation of multiple drug therapy (MDT) for leprosy has significantly reduced the global cases of leprosy. Currently, only a few endemic countries remain where relatively high number of cases persists. Despite global reduction of leprosy and the concomitant decrease in human reservoirs, leprosy transmission and incidence have not declined as expected, suggesting a possible extra-human or environmental source of the bacilli. In the current study, we demonstrate that M. leprae can survive long-term within cysts of common environmental free-living amoebae. M. leprae residing in amoebal cysts for over 30 days remain fully capable of transferring disease to mouse footpads and retain viability phenotypes after several months residence within amoebal cysts. It is hypothesized that these protozoa provide an intracellular refuge for M. leprae in environments for which they would otherwise seem ill suited. Traits allowing bacilli to survive in macrophages may likely be acquired via an evolutionary response against predation by amoebae. The results from this work suggest alternative non-human reservoirs for M. leprae exist fostering further study to determine the role of amoebae in the transmission of this Mycobacterium to humans.
doi:10.1371/journal.pntd.0003405
PMCID: PMC4270725  PMID: 25521850
3.  Phylogenomics of Brazilian epidemic isolates of Mycobacterium abscessus subsp. bolletii reveals relationships of global outbreak strains 
Rapidly growing, non-tuberculous mycobacteria (NTM) in the Mycobacterium abscessus (MAB) species are emerging pathogens that cause various diseases including skin and respiratory infections. The species has undergone recent taxonomic nomenclature refinement, and is currently recognized as two subspecies, M. abscessus subsp. abscessus (MAB-A) and M. abscessus subsp. bolletii (MAB-B). The recently reported outbreaks of MAB-B in surgical patients in Brazil from 2004 to 2009 and in cystic fibrosis patients in the United Kingdom (UK) in 2006 to 2012 underscore the need to investigate the genetic diversity of clinical MAB strains. To this end, we sequenced the genomes of two Brazilian MAB-B epidemic isolates (CRM-0019 and CRM-0020) derived from an outbreak of skin infections in Rio de Janeiro, two unrelated MAB strains from patients with pulmonary infections in the United States (US) (NJH8 and NJH11) and one type MAB-B strain (CCUG 48898) and compared them to 25 publically available genomes of globally diverse MAB strains. Genome-wide analyses of 27,598 core genome single nucleotide polymorphisms (SNPs) revealed that the two Brazilian derived CRM strains are nearly indistinguishable from one another and are more closely related to UK outbreak isolates infecting CF patients than to strains from the US, Malaysia or France. Comparative genomic analyses of six closely related outbreak strains revealed geographic-specific large-scale insertion/deletion variation that corresponds to bacteriophage insertions and recombination hotspots. Our study integrates new genome sequence data with existing genomic information to explore the global diversity of infectious M. abscessus isolates and to compare clinically relevant outbreak strains from different continents.
doi:10.1016/j.meegid.2013.09.012
PMCID: PMC3853122  PMID: 24055961
Mycobacterium abscessus; comparative genomics; nontuberculous mycobacteria; NTM; bioinformatics
4.  Liver Stiffness Increases Acutely During Sickle Cell Vaso-Occlusive Crisis 
American journal of hematology  2013;88(11):10.1002/ajh.23532.
Background & Aims
Acute vaso-occlusive crisis (VOC) in sickle cell disease (SCD) is an important cause of end-organ damage. It is estimated that 10–39% of VOC occurs with hepatic involvement. Current assessments of hepatic involvement during VOC are unsatisfactory. We investigated transient elastography (TE) as a marker of hepatic involvement, its relationship with histology, and biochemical markers during VOC.
Methods
SCD patients were evaluated with biochemical markers and TE at steady-state and during VOC. Change in TE and biochemical markers were correlated to length of hospital stay. When available, liver biopsy and tricuspid regurgitation velocity (TRV) at steady-state were correlated with TE.
Results
23 patients were evaluated (mean age=34.3 years, standard deviation=7.96). In 15 patients with liver biopsies, TE correlated with fibrosis (p=0.01) and TRV (p=0.0063), but not hepatic iron. Hemolysis biomarkers changed during VOC (p<0.022), but not alanine aminotransferase (ALT). Paired comparison of TE at steady-state and during VOC showed an increased from 6.2 to 12.3 kPa (p=0.0029). Increasing TE during VOC associated with increasing ALT and alkaline phosphatase (p=0.0088 and 0.0099, respectively). At steady-state, increasing inflammation on biopsy (p=0.0037) and TRV (p=0.0075) correlated with increasing TE during VOC. Increased hospital stay was associated with higher ALT (p=0.041), lower albumin (p=0.046), hemoglobin/hematocrit (p<0.0021) but not TE.
Conclusions
TE may identify patients with hepatic involvement during VOC independent of biochemical measures. Increase in TE may reflect both hepatic passive congestion and hepatic involvement during VOC. TE may serve as a physiological biomarker for hepatic features of VOC.
doi:10.1002/ajh.23532
PMCID: PMC3808506  PMID: 23828202
Transient Elastography; Sickle Cell Disease; Vaso-Occlusive Crisis; Hepatic Crisis; Liver Stiffness
5.  Mycobacterium tuberculosis Lipoprotein LprG Binds Lipoarabinomannan and Determines Its Cell Envelope Localization to Control Phagolysosomal Fusion 
PLoS Pathogens  2014;10(10):e1004471.
Mycobacterium tuberculosis (Mtb) virulence is decreased by genetic deletion of the lipoprotein LprG, but the function of LprG remains unclear. We report that LprG expressed in Mtb binds to lipoglycans, such as lipoarabinomannan (LAM), that mediate Mtb immune evasion. Lipoglycan binding to LprG was dependent on both insertion of lipoglycan acyl chains into a hydrophobic pocket on LprG and a novel contribution of lipoglycan polysaccharide components outside of this pocket. An lprG null mutant (Mtb ΔlprG) had lower levels of surface-exposed LAM, revealing a novel role for LprG in determining the distribution of components in the Mtb cell envelope. Furthermore, this mutant failed to inhibit phagosome-lysosome fusion, an immune evasion strategy mediated by LAM. We propose that LprG binding to LAM facilitates its transfer from the plasma membrane into the cell envelope, increasing surface-exposed LAM, enhancing cell envelope integrity, allowing inhibition of phagosome-lysosome fusion and enhancing Mtb survival in macrophages.
Author Summary
The causative agent of tuberculosis, Mycobacterium tuberculosis (Mtb), persists in phagosomes inside infected macrophages. Mtb expresses lipoarabinomannan (LAM), which inhibits fusion of phagosomes with lysosomes as a means for Mtb to evade host defense. LAM is present in the cell envelope, which surrounds Mtb and interfaces with the host, but its localization remains unclear. We show that LprG, an Mtb lipoprotein, binds LAM and controls its distribution in the cell envelope. A mutant strain of Mtb that lacks LprG has less LAM at the surface of the cell envelope. This decreases LAM-mediated inhibition of phagosome-lysosome fusion, thereby impairing an immune evasion mechanism. We propose that LprG facilitates transfer of LAM from the plasma membrane into the cell envelope, enhancing its interaction with the host and ability to regulate host defense. Our results reveal mechanisms that determine bacterial cell envelope function and influence host-pathogen interactions and pathogen evasion of host defense.
doi:10.1371/journal.ppat.1004471
PMCID: PMC4214796  PMID: 25356793
6.  LprG-Mediated Surface Expression of Lipoarabinomannan Is Essential for Virulence of Mycobacterium tuberculosis 
PLoS Pathogens  2014;10(9):e1004376.
Mycobacterium tuberculosis employs various virulence strategies to subvert host immune responses in order to persist and cause disease. Interaction of M. tuberculosis with mannose receptor on macrophages via surface-exposed lipoarabinomannan (LAM) is believed to be critical for cell entry, inhibition of phagosome-lysosome fusion, and intracellular survival, but in vivo evidence is lacking. LprG, a cell envelope lipoprotein that is essential for virulence of M. tuberculosis, has been shown to bind to the acyl groups of lipoglycans but the role of LprG in LAM biosynthesis and localization remains unknown. Using an M. tuberculosis lprG mutant, we show that LprG is essential for normal surface expression of LAM and virulence of M. tuberculosis attributed to LAM. The lprG mutant had a normal quantity of LAM in the cell envelope, but its surface was altered and showed reduced expression of surface-exposed LAM. Functionally, the lprG mutant was defective for macrophage entry and inhibition of phagosome-lysosome fusion, was attenuated in macrophages, and was killed in the mouse lung with the onset of adaptive immunity. This study identifies the role of LprG in surface-exposed LAM expression and provides in vivo evidence for the essential role surface LAM plays in M. tuberculosis virulence. Findings have translational implications for therapy and vaccine development.
Author Summary
Mycobacterium tuberculosis is among the leading infectious causes of human death. A better understanding of its virulence mechanisms is needed to facilitate development of novel therapeutics and a preventative vaccine. Lipoarabinomannan (LAM), an abundant surface-exposed lipoglycan, is believed to be a critical virulence determinant for intracellular survival and latency of M. tuberculosis. In vitro experiments with purified LAM have led to a model in which surface-exposed LAM binds to macrophage mannose receptor and facilitates bacterium entry, inhibition of phagosome-lysosome fusion, and modulation of innate immune responses. However, confirmation of these findings in vivo has not been possible due to the essentiality of genes involved in the LAM biosynthetic pathway. It was recently shown that LprG, a cell envelope lipoprotein, binds to the acyl groups of lipoglycan, but the role of LprG in LAM biosynthesis and localization remains unknown. Here, using an M. tuberculosis lprG mutant and a novel cell-imprinting assay, we show that LprG is essential for normal surface expression of LAM and virulence of M. tuberculosis attributed to LAM. Our study provides new insights into the mechanism of surface expression of LAM and confirms the essential role surface LAM serves in pathogenesis of M. tuberculosis.
doi:10.1371/journal.ppat.1004376
PMCID: PMC4169494  PMID: 25232742
7.  Biosynthesis and Translocation of Unsulfated Acyltrehaloses in Mycobacterium tuberculosis* 
The Journal of Biological Chemistry  2014;289(40):27952-27965.
Background: The biogenesis of 2,3-diacyltrehaloses (DAT) and penta-acyltrehaloses (PAT) found in the outer membrane of Mycobacterium tuberculosis is ill defined.
Results: DAT synthesis is cytosolic. Chp2-mediated transesterification reactions between DAT substrates yield PAT on the periplasmic face of the membrane.
Conclusion: DAT and PAT biosynthesis is topologically split across the membrane.
Significance: DAT and PAT biosynthesis and transport are coupled and dependent on the MmpL10 transporter.
A number of species-specific polymethyl-branched fatty acid-containing trehalose esters populate the outer membrane of Mycobacterium tuberculosis. Among them, 2,3-diacyltrehaloses (DAT) and penta-acyltrehaloses (PAT) not only play a structural role in the cell envelope but also contribute to the ability of M. tuberculosis to multiply and persist in the infected host, promoting the intracellular survival of the bacterium and modulating host immune responses. The nature of the machinery, topology, and sequential order of the reactions leading to the biosynthesis, assembly, and export of these complex glycolipids to the cell surface are the object of the present study. Our genetic and biochemical evidence corroborates a model wherein the biosynthesis and translocation of DAT and PAT to the periplasmic space are coupled and topologically split across the plasma membrane. The formation of DAT occurs on the cytosolic face of the plasma membrane through the action of PapA3, FadD21, and Pks3/4; that of PAT occurs on the periplasmic face via transesterification reactions between DAT substrates catalyzed by the acyltransferase Chp2 (Rv1184c). The integral membrane transporter MmpL10 is essential for DAT to reach the cell surface, and its presence in the membrane is required for Chp2 to be active. Disruption of mmpL10 or chp2 leads to an important build-up of DAT inside the cells and to the formation of a novel form of unsulfated acyltrehalose esterified with polymethyl-branched fatty acids normally found in sulfolipids that is translocated to the cell surface.
doi:10.1074/jbc.M114.581199
PMCID: PMC4183827  PMID: 25124040
Glycolipid; Mycobacteria; Polyketide; Transporter; Tuberculosis; Chp2; MmpL10; Acyltransferase; Acyltrehalose
9.  RND transporters protect Corynebacterium glutamicum from antibiotics by assembling the outer membrane 
MicrobiologyOpen  2014;3(4):484-496.
Corynebacterium–Mycobacterium–Nocardia (CMN) group are the causative agents of a broad spectrum of diseases in humans. A distinctive feature of these Gram-positive bacteria is the presence of an outer membrane of unique structure and composition. Recently, resistance–nodulation–division (RND) transporters (nicknamed MmpLs, Mycobacterial membrane protein Large) have emerged as major contributors to the biogenesis of the outer membranes in mycobacteria and as promising drug targets. In this study, we investigated the role of RND transporters in the physiology of Corynebacterium glutamicum and analyzed properties of these proteins. Our results show that in contrast to Gram-negative species, in which RND transporters actively extrude antibiotics from cells, in C. glutamicum and relatives these transporters protect cells from antibiotics by playing essential roles in the biogenesis of the low-permeability barrier of the outer membrane. Conditional C. glutamicum mutants lacking RND proteins and with the controlled expression of either NCgl2769 (CmpL1) or NCgl0228 (CmpL4) are hypersusceptible to multiple antibiotics, have growth deficiencies in minimal medium and accumulate intracellularly trehalose monocorynomycolates, free corynomycolates, and the previously uncharacterized corynomycolate-containing lipid. Our results also suggest that similar to other RND transporters, Corynebacterial membrane proteins Large (CmpLs) functions are dependent on a proton-motive force.
doi:10.1002/mbo3.182
PMCID: PMC4287177  PMID: 24942069
Cell envelope; membrane transport; resistance–nodulation–division family
10.  Characterization of Field Loss Based on Microperimetry Is Predictive of Face Recognition Difficulties 
Purpose.
To determine how visual field loss as assessed by microperimetry is correlated with deficits in face recognition.
Methods.
Twelve patients (age range, 26–70 years) with impaired visual sensitivity in the central visual field caused by a variety of pathologies and 12 normally sighted controls (control subject [CS] group; age range, 20–68 years) performed a face recognition task for blurred and unblurred faces. For patients, we assessed central visual field loss using microperimetry, fixation stability, Pelli-Robson contrast sensitivity, and letter acuity.
Results.
Patients were divided into two groups by microperimetry: a low vision (LV) group (n = 8) had impaired sensitivity at the anatomical fovea and/or poor fixation stability, whereas a low vision that excluded the fovea (LV:F) group (n = 4) was characterized by at least some residual foveal sensitivity but insensitivity in other retinal regions. The LV group performed worse than the other groups at all blur levels, whereas the performance of the LV:F group was not credibly different from that of the CS group. The performance of the CS and LV:F groups deteriorated as blur increased, whereas the LV group showed consistently poor performance regardless of blur. Visual acuity and fixation stability were correlated with face recognition performance.
Conclusions.
Persons diagnosed as having disease affecting the central visual field can recognize faces as well as persons with no visual disease provided that they have residual sensitivity in the anatomical fovea and show stable fixation patterns. Performance in this task is limited by the upper resolution of nonfoveal vision or image blur, whichever is worse.
Patients with central visual field loss and control subjects recognized faces in a 10-alternative forced-choice task. Patients with intact foveal sensitivity (assessed by microperimetry) performed similarly to controls; patients with foveal loss performed poorly. Perimetry is predictive of face recognition difficulty.
doi:10.1167/iovs.13-12420
PMCID: PMC3885139  PMID: 24302589
face recognition; central vision loss; low vision; perimetry; Bayesian inference
11.  Mechanism of inhibition of Mycobacterium tuberculosis antigen 85 by ebselen 
Nature communications  2013;4:2748.
The increasing prevalence of drug-resistant tuberculosis highlights the need for identifying new antitubercular drugs that can treat these infections. The antigen 85 (Ag85) complex has emerged as an intriguing mycobacterial drug target due to its central role in synthesizing major components of the inner and outer leaflets of the mycobacterial outer membrane. Here we identify ebselen as a potent inhibitor of the Mycobacterium tuberculosis Ag85 complex. Mass spectrometry data show that ebselen binds covalently to a cysteine residue (C209) located near the Ag85C active site. The crystal structure of Ag85C in the presence of ebselen shows that C209 modification restructures the active site, thereby disrupting the hydrogen-bonded network within the active site that is essential for enzymatic activity. C209 mutations display marked decreases in enzymatic activity. These data suggest that compounds using this mechanism of action will strongly inhibit the Ag85 complex and minimize the selection of drug resistance.
doi:10.1038/ncomms3748
PMCID: PMC4049535  PMID: 24193546
12.  Subfractionation and analysis of the cell envelope (lipo)polysaccharides of Mycobacterium tuberculosis 
The cell envelope of Mycobacterium tuberculosis, the causative agent of tuberculosis in humans, is the source of carbohydrates of exceptional structure which play essential roles in the physiology of the bacterium and in its interactions with the host during infection. Much of what is known about their biosynthesis was derived from the phenotypic analysis of knock-out or conditional knock-out mutants of Mycobacteria generated by random or specific insertional mutagenesis. Here, we describe the current techniques used to subfractionate M. tuberculosis cells and investigate major quantitative and qualitative changes in their cell envelope (lipo)polysaccharides.
doi:10.1007/978-1-62703-245-2_19
PMCID: PMC4041545  PMID: 23299743
Mycobacterium tuberculosis; arabinogalactan; lipoarabinomannan; lipomannan; glucan; capsule
13.  Assessment of minimally invasive surgical skills of pre-medical students: What can we learn from future learners? 
Objective
Knowledge of baseline laparoscopic and robotic surgical skills of future learners is essential to develop teaching strategies that best fit them. The objectives of this study are to determine baseline laparoscopic and robotic skills of high school and college students and compare them to those of current obstetrics and gynecology residents.
Material and Methods
A cross-sectional (Class II-2) pilot study. Laparoscopic and robotic surgical skills of college and high (secondary) school students were evaluated using simulators and compared to those of obstetrics and gynecology residents. In addition, questionnaire data were collected regarding video game playing and computer use.
Results
A total of 17 students, both high school (n=9) and college (n=8), in addition to 11 residents, completed the study. Overall, students performed comparably to the residents in simple exercises (p>.05). However, students took significantly longer time to complete complex exercises (p=.001). Finally, students played video games significantly more than residents (p<.001).
Conclusion
Future learners may have a different background skill set. This difference may be related to improved hand-eye coordination, possibly due to playing video games. The results of this pilot study should spur more research into surgical teaching strategies.
doi:10.5152/jtgga.2014.0044
PMCID: PMC4072552  PMID: 24976769
Simulation; education; robotic surgery; laparoscopy; video games
14.  Inactivation of Fructose-1,6-Bisphosphate Aldolase Prevents Optimal Co-catabolism of Glycolytic and Gluconeogenic Carbon Substrates in Mycobacterium tuberculosis 
PLoS Pathogens  2014;10(5):e1004144.
Metabolic pathways used by Mycobacterium tuberculosis (Mtb) to establish and maintain infections are important for our understanding of pathogenesis and the development of new chemotherapies. To investigate the role of fructose-1,6-bisphosphate aldolase (FBA), we engineered an Mtb strain in which FBA levels were regulated by anhydrotetracycline. Depletion of FBA resulted in clearance of Mtb in both the acute and chronic phases of infection in vivo, and loss of viability in vitro when cultured on single carbon sources. Consistent with prior reports of Mtb's ability to co-catabolize multiple carbon sources, this in vitro essentiality could be overcome when cultured on mixtures of glycolytic and gluconeogenic carbon sources, enabling generation of an fba knockout (Δfba). In vitro studies of Δfba however revealed that lack of FBA could only be compensated for by a specific balance of glucose and butyrate in which growth and metabolism of butyrate were determined by Mtb's ability to co-catabolize glucose. These data thus not only evaluate FBA as a potential drug target in both replicating and persistent Mtb, but also expand our understanding of the multiplicity of in vitro conditions that define the essentiality of Mtb's FBA in vivo.
Author Summary
The development of new chemotherapies targeting Mycobacterium tuberculosis (Mtb) will benefit from genetic evaluation of potential drug targets and a better understanding of the pathways required by Mtb to establish and maintain chronic infections. We employed a genetic approach to investigate the essentiality of fructose-1,6-bisphosphate aldolase (FBA) for growth and survival of Mtb in vitro and in mice. A conditional fba mutant revealed that Mtb requires FBA for growth in the acute phase and for survival in the chronic phase of mouse infections. In vitro essentiality of fba was strictly condition-dependent. An FBA deletion mutant (Δfba) required a balanced combination of carbon substrates entering metabolism above and below the FBA-catalyzed reaction for growth and died in media with single carbon sources and in mouse lungs. Death of Δfba in vitro was associated with the perturbation of intracellular metabolites. These studies highlight how a conditional fba mutant helped identify conditions in which FBA is dispensable for growth of Mtb, evaluate FBA as a potential target for eliminating persistent bacilli and offer insight into metabolic regulation of carbon co-catabolism in Mtb.
doi:10.1371/journal.ppat.1004144
PMCID: PMC4031216  PMID: 24851864
15.  Design, Synthesis and Anti-tuberculosis Activity of 1-Adamantyl-3-heteroaryl Ureas with Improved in vitro Pharmacokinetic Properties 
Bioorganic & medicinal chemistry  2013;21(9):2587-2599.
Out of the prominent global ailments, tuberculosis (TB) is still one of the leading causes of death worldwide due to infectious disease. Development of new drugs that shorten the current tuberculosis treatment time and have activity against drug resistant strains is of utmost importance. Towards these goals we have focused our efforts on developing novel anti-TB compounds with the general structure of 1-adamantyl-3-phenyl urea. This series is active against Mycobacteria and previous lead compounds were found to inhibit the membrane transporter MmpL3, the protein responsible for mycolic acid transport across the plasma membrane. However, these compounds suffered from poor in vitro pharmacokinetic (PK) profiles and they have a similar structure/SAR to inhibitors of human soluble epoxide hydrolase (SEH) enzymes. Therefore, in this study the further optimization of this compound class was driven by three factors: 1) to increase selectivity for anti-TB activity over human sEH activity, 2) to optimize PK profiles including solubility and 3) to maintain target inhibition. A new series of 1-adamantyl-3-heteroaryl ureas was designed and synthesized replacing the phenyl substituent of the original series with pyridines, pyrimidines, triazines, oxazoles, isoxazoles, oxadiazoles and pyrazoles. This study produced lead oxadiazole and pyrazole substituted adamantyl ureas with improved in vitro PK profiles, increased selectivity and good anti-TB potencies with sub µg/mL minimum inhibitory concentrations.
doi:10.1016/j.bmc.2013.02.028
PMCID: PMC3780361  PMID: 23498915
16.  Progress in targeting cell envelope biogenesis in Mycobacterium tuberculosis 
Future microbiology  2013;8(7):10.2217/fmb.13.52.
Most of the newly discovered compounds showing promise for the treatment of TB, notably multidrug-resistant TB, inhibit aspects of Mycobacterium tuberculosis cell envelope metabolism. This review reflects on the evolution of the knowledge that many of the front-line and emerging products inhibit aspects of cell envelope metabolism and in the process are bactericidal not only against actively replicating M. tuberculosis, but contrary to earlier impressions, are effective against latent forms of the disease. While mycolic acid and arabinogalactan synthesis are still primary targets of existing and new drugs, peptidoglycan synthesis, transport mechanisms and the synthesis of the decaprenyl-phosphate carrier lipid all show considerable promise as targets for new products, older drugs and new combinations. The advantages of whole cell- versus target-based screening in the perpetual search for new targets and products to counter multidrug-resistant TB are discussed.
doi:10.2217/fmb.13.52
PMCID: PMC3867987  PMID: 23841633
antibiotic; arabinogalactan; cell envelope; Mycobacterium; mycolic acids; peptidoglycan; tuberculosis
17.  Gene Replacement in Mycobacterium chelonae: Application to the Construction of Porin Knock-Out Mutants 
PLoS ONE  2014;9(4):e94951.
Mycobacterium chelonae is a rapidly growing mycobacterial opportunistic pathogen closely related to Mycobacterium abscessus that causes cornea, skin and soft tissue infections in humans. Although M. chelonae and the emerging mycobacterial pathogen M. abscessus have long been considered to belong to the same species, these two microorganisms considerably differ in terms of optimum growth temperature, drug susceptibility, pathogenicity and the types of infection they cause. The whole genome sequencing of clinical isolates of M. chelonae and M. abscessus is opening the way to comparative studies aimed at understanding the biology of these pathogens and elucidating the molecular bases of their pathogenicity and biocide resistance. Key to the validation of the numerous hypotheses that this approach will raise, however, is the availability of genetic tools allowing for the expression and targeted mutagenesis of genes in these species. While homologous recombination systems have recently been described for M. abscessus, genetic tools are lacking for M. chelonae. We here show that two different allelic replacement methods, one based on mycobacteriophage-encoded recombinases and the other on a temperature-sensitive plasmid harboring the counterselectable marker sacB, can be used to efficiently disrupt genes in this species. Knock-out mutants for each of the three porin genes of M. chelonae ATCC 35752 were constructed using both methodologies, one of which displays a significantly reduced glucose uptake rate consistent with decreased porin expression.
doi:10.1371/journal.pone.0094951
PMCID: PMC3989263  PMID: 24739882
18.  High-level Relatedness among Mycobacterium abscessus subsp. massiliense Strains from Widely Separated Outbreaks 
Emerging Infectious Diseases  2014;20(3):364-371.
Three recently sequenced strains isolated from patients during an outbreak of Mycobacterium abscessus subsp. massiliense infections at a cystic fibrosis center in the United States were compared with 6 strains from an outbreak at a cystic fibrosis center in the United Kingdom and worldwide strains. Strains from the 2 cystic fibrosis outbreaks showed high-level relatedness with each other and major-level relatedness with strains that caused soft tissue infections during an epidemic in Brazil. We identified unique single-nucleotide polymorphisms in cystic fibrosis and soft tissue outbreak strains, separate single-nucleotide polymorphisms only in cystic fibrosis outbreak strains, and unique genomic traits for each subset of isolates. Our findings highlight the necessity of identifying M. abscessus to the subspecies level and screening all cystic fibrosis isolates for relatedness to these outbreak strains. We propose 2 diagnostic strategies that use partial sequencing of rpoB and secA1 genes and a multilocus sequence typing protocol.
doi:10.3201/eid2003.131106
PMCID: PMC3944860  PMID: 24565502
Mycobacterium abscessus; Mycobacterium abscessus subsp. massiliense; bacteria; tuberculosis and other mycobacteria; strains; cystic fibrosis; relatedness; outbreaks; geographically distant outbreaks; United States; United Kingdom
19.  Guidance on Management of Asymptomatic Neonates Born to Women With Active Genital Herpes Lesions 
Pediatrics  2013;131(2):e635-e646.
Herpes simplex virus (HSV) infection of the neonate is uncommon, but genital herpes infections in adults are very common. Thus, although treating an infant with neonatal herpes is a relatively rare occurrence, managing infants potentially exposed to HSV at the time of delivery occurs more frequently. The risk of transmitting HSV to an infant during delivery is determined in part by the mother’s previous immunity to HSV. Women with primary genital HSV infections who are shedding HSV at delivery are 10 to 30 times more likely to transmit the virus to their newborn infants than are women with recurrent HSV infection who are shedding virus at delivery. With the availability of commercial serological tests that reliably can distinguish type-specific HSV antibodies, it is now possible to determine the type of maternal infection and, thus, further refine management of infants delivered to women who have active genital HSV lesions. The management algorithm presented herein uses both serological and virological studies to determine the risk of HSV transmission to the neonate who is delivered to a mother with active herpetic genital lesions and tailors management accordingly. The algorithm does not address the approach to asymptomatic neonates delivered to women with a history of genital herpes but no active lesions at delivery.
doi:10.1542/peds.2012-3216
PMCID: PMC3557411  PMID: 23359576
newborn; herpes simplex virus; acyclovir; pregnancy
20.  Feasibility of a Web-Based Survey of Hallucinations and Assessment of Visual Function in Patients With Parkinson’s Disease 
Background
Patients with Parkinson’s disease (PD) experience visual hallucinations, which may be related to decreased contrast sensitivity (ie, the ability to discern shades of grey).
Objective
The objective of this study was to investigate if an online research platform can be used to survey patients with Parkinson’s disease regarding visual hallucinations, and also be used to assess visual contrast perception.
Methods
From the online patient community, PatientsLikeMe, 964 members were invited via email to participate in this study. Participants completed a modified version of the University of Miami Parkinson’s disease hallucinations questionnaire and an online vision test.
Results
The study was completed by 27.9% (269/964) of those who were invited: 56.9% of this group had PD (153/269) and 43.1% (116/269) were non-Parkinson’s controls. Hallucinations were reported by 18.3% (28/153) of the Parkinson’s group. Although 10 subjects (9%) in the control group reported experiencing hallucinations, only 2 of them actually described formed hallucinations. Participants with Parkinson’s disease with a mean of 1.75 (SD 0.35) and the control group with a mean of 1.85 (SD 0.36) showed relatively good contrast perception as measured with the online letter test (P=.07). People who reported hallucinations showed contrast sensitivity levels that did not differ from levels shown by people without hallucinations (P=.96), although there was a trend towards lower contrast sensitivity in hallucinators.
Conclusions
Although more Parkinson's responders reported visual hallucinations, a significant number of non-Parkinson's control group responders also reported visual hallucinations. The online survey method may have failed to distinguish between formed hallucinations, which are typical in Parkinson's disease, and non-formed hallucinations that have less diagnostic specificity. Multiple questions outlining the nature of the hallucinations are required. In a clinical interview, the specific nature of the hallucination would be further refined to rule out a vague description that does not indicate a true, formed visual hallucination. Contrary to previous literature, both groups showed relatively good contrast sensitivity, perhaps representing a ceiling effect or limitations of online testing conditions that are difficult to standardize. Steps can be taken in future trials to further standardize online visual function testing, to refine control group parameters and to take steps to rule out confounding variables such as comorbid disease that could be associated with hallucinations. Contacting subjects via an online health social network is a novel, cost-effective method of conducting vision research that allows large numbers of individuals to be contacted quickly, and refinement of questionnaires and visual function testing may allow more robust findings in future research.
doi:10.2196/ijmr.2744
PMCID: PMC3906694  PMID: 24394559
Parkinson’s disease; hallucinations; contrast sensitivity; Charles Bonnet Syndrome
21.  Trends in Adverse Reactions to Trimethoprim-Sulfamethoxazole 
Pediatrics  2013;131(1):e103-e108.
OBJECTIVE:
To examine temporal trends of adverse drug reactions (ADRs) associated with trimethoprim-sulfamethoxazole (TMP-SMX) use in children.
METHODS:
We performed a retrospective observational study to characterize TMP-SMX ADRs in children between 2000 and 2009. We completed a chart review at our institution by identifying children diagnosed with TMP-SMX ADRs. To compare local trends to comparable institutions, we estimated the frequency of hospitalizations for TMP-SMX ADRs at 25 tertiary pediatric hospitals utilizing the Pediatric Health Information System database. To determine whether changes in outpatient prescribing rates occurred, we used the National Ambulatory Medical Care Survey/National Hospital Ambulatory Medical Care Survey.
RESULTS:
At our institution, 109 children were diagnosed with a TMP-SMX ADR (5 cases from 2000 to 2004 as compared with 104 cases from 2005 to 2009). Fifty-eight percent had been treated for a skin and soft tissue infection (SSTI). A similar trend was observed nationally, where the incidence of TMP-SMX ADRs more than doubled from 2004 to 2009 at comparable pediatric hospitals (P < .001). Although national outpatient data revealed no change in overall TMP-SMX prescribing, the percentage of children prescribed TMP-SMX for SSTI sharply increased during the study period (0%–2% [2000-2004]; 9%–17% [2005–2009]).
CONCLUSIONS:
The majority of TMP-SMX ADRs at our institution occurred in conjunction with SSTI treatment. TMP-SMX ADRs have occurred more frequently coincident with increased prescribing for SSTI. Increased usage alone may explain the increasing trend of TMP-SMX ADRs in children; however drug–disease interaction may play a role and requires further investigation.
doi:10.1542/peds.2012-1619
PMCID: PMC3529952  PMID: 23209098
adverse drug reaction; trimethoprim-sulfamethoxazole; skin and soft tissue infections; pediatrics; physician practice patterns
22.  Misleading hepatitis B testing in the setting of intravenous immunoglobulin 
F1000Research  2013;2:249.
Intravenous immunoglobulin (IVIG) is commonly used for a wide range of diagnoses, by multiple pediatric subspecialists. We report two cases of hepatitis B screening results post IVIG infusion, where positive anti-Hepatitis B core antigen serology tests indicated possible occult hepatitis infection, leading to a delay in care. However, serial antibody testing showed results consistent with the passive transfer of antibodies.
doi:10.12688/f1000research.2-249.v1
PMCID: PMC4103488  PMID: 25075281
23.  Regulation of Mycolactone, the Mycobacterium ulcerans Toxin, Depends on Nutrient Source 
Background
Mycobacterium ulcerans, a slow-growing environmental bacterium, is the etiologic agent of Buruli ulcer, a necrotic skin disease. Skin lesions are caused by mycolactone, the main virulence factor of M. ulcerans, with dermonecrotic (destruction of the skin and soft tissues) and immunosuppressive activities. This toxin is secreted in vesicles that enhance its biological activities. Nowadays, it is well established that the main reservoir of the bacilli is localized in the aquatic environment where the bacillus may be able to colonize different niches. Here we report that plant polysaccharides stimulate M. ulcerans growth and are implicated in toxin synthesis regulation.
Methodology/Principal Findings
In this study, by selecting various algal components, we have identified plant-specific carbohydrates, particularly glucose polymers, capable of stimulating M. ulcerans growth in vitro. Furthermore, we underscored for the first time culture conditions under which the polyketide toxin mycolactone, the sole virulence factor of M. ulcerans identified to date, is down-regulated. Using a quantitative proteomic approach and analyzing transcript levels by RT-qPCR, we demonstrated that its regulation is not at the transcriptional or translational levels but must involve another type of regulation. M. ulcerans produces membrane vesicles, as other mycobacterial species, in which are the mycolactone is concentrated. By transmission electron microscopy, we observed that the production of vesicles is independent from the toxin production. Concomitant with this observed decrease in mycolactone production, the production of mycobacterial siderophores known as mycobactins was enhanced.
Conclusions/Significance
This work is the first step in the identification of the mechanisms involved in mycolactone regulation and paves the way for the discovery of putative new drug targets in the future.
Author Summary
Mycolactone, a polyketide cytotoxic toxin, is the key virulence factor responsible for large skin ulcers in Buruli ulcer. This disease, mainly occurring in humid tropical zones, especially in West African countries, is due to infection by Mycobacterium ulcerans, a slow-growing environmental mycobacterium. The toxin has destructive effects on the skin, soft tissues and bones. In this study, we brought out for the first time that addition of specific carbohydrate to culture medium induces the down-regulation of the toxin. Furthermore, this decrease in toxin production is correlated with the activation of the iron acquisition pathway, especially by siderophore production. These results show that M. ulcerans adapts its metabolism to culture conditions, which probably reflect its adaptation in its natural habitats. This work is the first step in toxin regulation understanding which is a key issue for a better comprehension of Buruli ulcer physiopathology and the identification of putative therapeutic targets.
doi:10.1371/journal.pntd.0002502
PMCID: PMC3828164  PMID: 24244764
24.  Aldehyde-Resistant Mycobacteria Associated with the Use of Endoscope Reprocessing Systems 
Bacteria can develop resistance to antibiotics, but less is known about their ability to increase resistance to chemical disinfectants. This study randomly sampled three AERs in the USA using aldehydes for endoscope disinfection. Bacterial contamination was found post-disinfection in all AERs and some mycobacteria isolated demonstrated significant resistance to glutaraldehyde and OPA disinfectants. Bacteria can survive aldehyde-based disinfection and may pose a cross-contamination risk to patients.
doi:10.1016/j.ajic.2011.11.004
PMCID: PMC3523318  PMID: 22325730
25.  Cooccurrence of Free-Living Amoebae and Nontuberculous Mycobacteria in Hospital Water Networks, and Preferential Growth of Mycobacterium avium in Acanthamoeba lenticulata 
Applied and Environmental Microbiology  2013;79(10):3185-3192.
The incidence of lung and other diseases due to nontuberculous mycobacteria (NTM) is increasing. NTM sources include potable water, especially in households where NTM populate pipes, taps, and showerheads. NTM share habitats with free-living amoebae (FLA) and can grow in FLA as parasites or as endosymbionts. FLA containing NTM may form cysts that protect mycobacteria from disinfectants and antibiotics. We first assessed the presence of FLA and NTM in water and biofilm samples collected from a hospital, confirming the high prevalence of NTM and FLA in potable water systems, particularly in biofilms. Acanthamoeba spp. (genotype T4) were mainly recovered (8/17), followed by Hartmannella vermiformis (7/17) as well as one isolate closely related to the genus Flamella and one isolate only distantly related to previously described species. Concerning mycobacteria, Mycobacterium gordonae was the most frequently found isolate (9/17), followed by Mycobacterium peregrinum (4/17), Mycobacterium chelonae (2/17), Mycobacterium mucogenicum (1/17), and Mycobacterium avium (1/17). The propensity of Mycobacterium avium hospital isolate H87 and M. avium collection strain 104 to survive and replicate within various FLA was also evaluated, demonstrating survival of both strains in all amoebal species tested but high replication rates only in Acanthamoeba lenticulata. As A. lenticulata was frequently recovered from environmental samples, including drinking water samples, these results could have important consequences for the ecology of M. avium in drinking water networks and the epidemiology of disease due to this species.
doi:10.1128/AEM.03823-12
PMCID: PMC3685249  PMID: 23475613

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